There is a fundamental gap in understanding how thymocyte clonal deletion and regulatory T cell (Treg) differentiation are coordinated during thymic T cell selection. Continued existence of this gap represents an important problem because, until it is filled, thymic T cell selection, which is central to immune regulation and tolerance, will remain largely incomprehensible. The objective of this application is to identify te specific molecular mechanisms by which dendritic cells (DCs) mediate clonal deletion and Treg differentiation in the thymus. Our central hypothesis is that thymic DCs coordinate Treg induction and clonal deletion by controlling surface turnover of peptide/MHCII complexes via regulated expression of membrane-associated ring finger CH1 (MARCH1) E3 ubiquitin ligase. This hypothesis has been formulated on the basis of preliminary data produced in the applicant's laboratory. The rationale for the proposed research is that once it is known how thymic DCs mediate clonal deletion and Treg development, novel strategies could be developed to exploit thymic DCs for immunotherapy aiming for the induction of antigen-specific T cell tolerance. Guided by strong preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Determine the role and extent to which MARCH1 contributes to Treg development in the thymus; 2) Identify the specific molecular mechanism by which MARCH1 mediates Treg development; and 3) Define the mechanism and function of heterogeneous MARCH1 expression among thymic DCs. Under the first aim, mice that are deficient in MARCH1 either entirely or DC-specific fashion will be examined for the development of antigen-specific Treg. Under the second aim, MHCII (K>R) knock-in mice in which MHCII cytoplasmic lysine (K) is replaced with arginine (R) and thus resistant to ubiquitination by MARCH1, will be examined to determine the specific role of MARCH1-mediated MHCII ubiquitination in Treg development. Under the third aim, MARCH1 regulation in thymic DCs and its functional role in clonal deletion will be determined by the approach of thymic DC transfer in combination with MARCH1 deregulation. Mouse strains and experimental skills required for successful completion of these aims are already on the applicants' hand. The approach is innovative, because it ablates physiologic regulation of MHCII-mediated antigen presentation in DCs and determines its consequence to T cell selection. The proposed research is significant, because it is expected to improve and vertically advance our understanding of T cell selection from conceptual understanding to mechanistically defined one. Ultimately, such understanding has the potential to be therapeutically exploited to develop antigen-specific tolerance for the treatment of hyper-immune disorders.